TGF-β type receptor cDNAs encoded products and uses therefor

ABSTRACT

DNA encoding TGF-β TYPE III receptor of mammalian origin, DNA encoding TGF-β type II receptor of mammalian origin, TGF-β type III receptor, TGF-β type II receptor and uses therefor.

This application is a division of application Ser. No. 08/311,703 filedSep. 23, 1994, now U.S. Pat. No. 6,010,872, which is a File WrapperContinuation of Ser. No. 07/786,063, filed Oct. 31, 1991 now abandoned.All of the above applications are incorporated herein by reference intheir entirety.

FUNDING

Work described herein was funded by National Cancer Institute Grant No.R35-CA39826; National Heart, Lung and Blood Institute Centers ofExcellence Grant HL-41484; the Damon Runyon-Walter Winchell CancerResearch Fund; National Institutes of Health predoctoral training grantnumber T 32 BM07287-16; and the American Cancer Society. The UnitedStates government has certain rights in the invention.

DESCRIPTION

Background

Transforming growth factor-beta (TGF-β) is a member of a family ofstructurally related cytokines that elicit a variety of responses,including growth, differentiation, and morphogenesis, in many differentcell types. (Roberts, A. B. and M. B. Sporn, In: Peptide Growth Factorsand Their Receptors, Springer-Verlag, Heidelberg, pp. 421-472 (1990);Massague, J., Annu. Rev. Cell. Biol. 6:597-641 (1990)) In vertebrates atleast five different forms of TGF-β, termed TGF-β1 to TGF-β5, have beenidentified; they all share a high degree (60%-80%) of amino-acidsequence identity. While TGF-β1 was initially characterized by itsability to induce anchorage-independent growth of normal rat kidneycells, its effects on most cell types are anti-mitogenic. (Altschul, S.F. et al., J. Mol. Biol. 215:403-410 (1990); Andres, J. L. et al., J.Cell. Biol. 109:3137-3145 (1989)) It is strongly growth-inhibitory formany types of cells, including both normal and transformed epithelial,endothelial, fibroblast, neuronal, lymphoid, and hematopoietic cells. Inaddition, TGF-β plays a central role in regulating the formation ofextracellular matrix and cell-matrix adhesion processes.

In spite of its widespread effects on cell phenotype and physiology,little is known about the biochemical mechanisms that enable TGF-βfamily members to elicit these varied responses. Three distincthigh-affinity cell-surface TGF-β-binding proteins, termed type I, II andIII, have been identified by incubating cells with radiolabelled TGF-β1,cross-linking bound TGF-β1 to cell surface molecules, and analyzing thelabelled complexes by polyacrylamide gel electrophoresis. (Massague, J.and B. Like, J. Biol. Chem. 260:2636-2645 (1985); Cheifetz, S. et al. J.Biol. Chem. 261:9972-9978 (1986).) The binding constants are about 5-50pM for the type I and II receptor and 30-300 pM for the type IIIreceptor. (Boyd, F. T. and J. Massague, J. Biol. Chem. 264:2272-2278(1989))

The type I and II receptors, of estimated 53 and 70-100 kilodaltons massrespectively, are N-glycosylated transmembrane proteins that are similarin many respects. Each of these receptors has a distinct affinity foreach member of the TGF-β family of ligands. (Boyd, F. T. and J.Massague, J. Biol. Chem. 264:2272-2278 (1989)) In contrast, the type IIIreceptor shows comparable affinities for all TGF-β isotypes; the typeIII receptor is the most abundant cell-surface receptor for TGF-β inmany cell lines (upwards of 200,000 per cell), and is an integralmembrane proteoglycan. It is heavily modified by glycosaminoglycan (GAG)groups, and migrates heterogeneously upon gel electrophoresis asproteins of 280 to 330 kilodaltons. When deglycosylated withheparitinase and chondrontinase, the protein core migrates as a 100-110kilodalton protein. The TGF-β binding site resides in this protein core,as non-glycosylated forms of this receptor that are produced in cellmutants defective in GAG synthesis are capable of ligand binding withaffinities comparable to those of the natural receptor. (Cheifetz, S.and J. Massague, J. Biol. Chem., 264:12025-12028 (1989) A variant formof type III receptor is secreted by some types of cells as a solublemolecule that apparently lacks a membrane anchor. This soluble speciesis found in low amounts in serum and in extracellular matrix.

The type III receptor, also called betaglycan, has a biological functiondistinct from that of the type I and II receptors. Some mutant mink lungepithelial cell (Mv1Lu) selected for loss of TGF-β responsiveness nolonger express type I receptors; others, similarly selected, loseexpression of both the type I and II receptors. However, all thesevariants continue to express the type III receptor. (Boyd, F. T. and J.Massague, J. Biol. Chem. 264:2272-2278 (1989); Laiho, M. et al., J.Biol. Chem. 265:18518-18524 (1990)) This has led to the proposal thattypes I and II receptors are signal-transducing molecules while the typeIII receptor, may subserve some other function, such as in concentratingligand before presentation to the bona fide signal-transducingreceptors. The secreted form of type III receptor, on the other hand,may act as a reservoir or clearance system for bioactive TGF-β.

Additional information about each of these TGF-β receptor types wouldenhance our understanding of their roles and make it possible, ifdesired, to alter their functions.

SUMMARY OF THE INVENTION

The present invention relates to isolation, sequencing andcharacterization of DNA encoding the TGF-β type III receptor ofmammalian origin and DNA encoding the TGF-β type II receptor ofmammalian origin. It also relates to the encoded TGF-β type III and typeII receptors, as well as to the soluble form of each; uses of thereceptor-encoding genes and of the receptors themselves; antibodiesspecific for TGF-β type III receptor and antibodies specific for TGF-βtype II receptor. In particular, it relates to DNA encoding the TGF-βtype III receptor of rat and human origin, DNA encoding the TGF-β typeII receptor of human origin and homologues of each.

The TGF-β receptor-encoding DNA of the present invention can be used-toidentify equivalent TGF-β receptor type III and type II genes from othersources, using, for example, known hybridization-based methods or thepolymerase chain reaction. The type III receptor gene, the type IIreceptor gene or their respective encoded products can be used to alterthe effects of TGF-β (e.g., by altering receptivity of cells to TGF-β orinterfering with binding of TGF-β to its receptor), such as its effectson cell proliferation or growth, cell adhesion and cell phenotype. Forexample, the TGF-β receptor type III gene, the TGF-β receptor type IIgene, or a truncated gene which encodes less than the entire receptor(e.g., soluble TGF-β type III receptor, soluble TGF-β type II receptoror the TGF-β type III or type II binding site) can be administered to anindividual in whom TGF-β effects are to be altered. Alternatively, theTGF-β type III receptor, the TGF-β type II receptor, a soluble formthereof (i.e., a form lacking the membrane anchor) or an active bindingsite of the TGF-β type III or the type II receptor can be administeredto an individual to alter the effects of TGF-β.

Because of the many roles TGF-β has in the body, availability of theTGF-β receptors described herein makes it possible to further assessTGF-β function and to alter (enhance or diminish) its effects.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C (SEQ ID NOS:5 and 6) are the DNA sequence and the translatedamino acid sequence of type III TGF-β1 receptor cDNA clone R3-OFF (fullinsert size 6 kb), in which the open reading frame with flankingsequences of the clone are shown. The transmembrane domain is indicatedby a single underline. Peptide sequences from purified type IIIreceptor, mentioned in text, that correspond to the derived sequence,are in italics and underlined. Potential N-linked glycosylation sitesare indicated by #, and extracellular cysteines by &. A consensusprotein kinase C phosphorylation site is indicated by $. The lastnon-vectorencoded amino acid of Clone R3-OF (2.9 kb) is indicated by @.Consensus proteoglycan attachment site is indicated by +++. Otherpotential glycosaminoglycan attachment sites are indicated by +. Theupstream in-frame stop codon (−42 to −44) is indicated by a wavy line.Signal peptide cleavage site predicted by vonHeijne's algorithm (vonHeijne, G., Nucl. Acid. Res. 14:4683-4690 (1986) is indicated by anarrow.

FIGS. 2A-2B (SEQ ID NO:7) are the nucleotide sequence of the full-lengthtype II TGF-β receptor cDNA clone 3FF isolated from a human HepG2 cellcDNA library (full insert size 5 kb). The cDNA has an open reading frameencoding a 567 amino acid residue protein.

FIG. 3 (SEQ ID NO:8) is the amino acid sequence of the full-length typeII TGF-β receptor.

DETAILED DESCRIPTION OF THE INVENTION

The subject invention is based on the isolation and sequencing of DNA ofvertebrate, particularly mammalian, origin which encodes TGF-β type IIIreceptor and DNA of mammalian origin which encodes TGF-β type IIreceptor, expression of the encoded products and characterization of theexpressed products. As described, a full-length cDNA which encodes TGF-βreceptor type III has been isolated from a cDNA library constructed froma rat vascular smooth muscle cell line and a full-length cDNA whichencodes TGF-β type II receptor has been isolated from a human cDNAlibrary. The human homologue of the type III gene has also been cloned.A deposit of human TGF-β type III cDNA in the plasmid pBSK has been madeunder the terms of the Budapest Treaty at the American Type CultureCollection (Oct. 21, 1991) 10801 University Boulevard, Manassas, Va.,20110-2209, under Accession Number 75127. All restrictions upon theavailability of the deposited material will be irrevocably removed upongranting of a U.S. patent based on the subject application.

Isolation and Characterization of TGF-β Type III Receptor

As described herein, two separate strategies were pursued for theisolation of the TCF-β type III receptor cDNA. In one approach,monoclonal antibodies were generated against the type III receptorprotein and used to purify the receptor, which was then subjected tomicrosequencing. (See Example 1) Microsequencing of several peptidesresulting from partial proteolysis of the purified receptor producedfour oligopeptide sequences, which were used to construct degenerateoligonucleotides. The degenerate oligonucleotides were used either asprimers in a cloning strategy using the polymerase chain reaction (PCR)or as probes in screening cDNA libraries. Although this strategy did notprove to be productive, the oligopeptide sequences were useful inverifying the identity of the receptor clones isolated by the secondstrategy.

In the second approach to isolating TGF-β receptor-encoding clones, anexpression cloning strategy was used in COS cells; direct visualizationof receptor positive cells was used to isolate receptor cDNAs. (SeeExample 2) In this approach, a cDNA library was constructed from A-10cells, a rat vascular smooth muscle cell line which expresses all threeTGF-β receptors (type I, II and III). COS cells transfected with cDNAcomponents of this library in a vector carrying the cytomegalovirus(CMV) transcriptional promoter and the SV40 origin of replication werescreened to identify cells expressing substantially higher than normallevels of TGF-β receptor. One transfectant expressing such high levelsof a TGF-β binding protein was identified and the original pool ofexpression constructs from which it was derived was split into subpools,which were subjected to a second round of screening. Two further roundsof sib-selection resulted in isolation of one cDNA clone (R3-OF) with a2.9 kb insert which induced high levels of TGF-β-binding proteins inapproximately 10% of cells into which it was introduced. The specificityof the TGF-β binding was validated by showing that addition of a200-fold excess unlabeled competitor TGF-β1 strongly reduced binding of125 I-TCF-β to transfected cells.

The R3-OF cDNA encoded an open reading frame of 817 amino acid residues,but did not contain a stop codon. R3-OF was used as a probe to isolate afull-length cDNA from a rat 208F library. The resulting clone, R3-OFF,is 6 kb in length and encodes a protein of 853 amino acids, which iscolinear with clone R3-OF. The nucleotide sequence of R3-OFF is shown inFIGS. 1A-1C, along with the translated amino acid sequence.

Characterization of the receptor encoded by R3-OFF was carried out, asdescribed in Example 3. Results showed three distinct TGF-β bindingprotein species of TGF-β on the surface of mock-transfected COS cells,which is in accord with results reported by others. (Massague, J. etal., Ann. NY Acad. Sci. 593:59-72 (1990)). These included the two lowermolecular weight type I and II receptors (65 and 85 kD) and the highermolecular weight type III proteoglycan, which migrates as a diffuse bandof 280-330 kd. Enzymatic removal of the proteoglycan yielded a coreprotein of approximately 100 kd. Binding to all three receptor types isspecific in that it was competed by 200-fold excess of unlabeled TGF-β1.

Transfecting the isolated cDNA caused a two-fold increase in expressionof the type III receptor. When a cell lysate derived from COS cellstransfected with clone R3-OFF was treated with deglycosylating enzymes,the heterogeneous 280-330 kd band was converted to a protein core whichco-migrates with the type III protein core seen in parental A10 cells.Importantly, the recombinant protein core migrated differently from theendogenous COS cell type III protein core.

These observations were confirmed and extended using stably transfectedcells expressing the type III cDNA. L6 rat skeleton muscle myoblasts donot express any detectable type III mRNA and no endogeneous surface typeIII receptor (Massague et al., 1986; Segarini et al., 1989). These cellswere transfected with the isolated cDNA in the vector pcDNA-neo. Cellclones stably expressing this clone in both the forward and reverseorientations with respect to the CMV promoter were isolated and analyzedby ligand binding assay.

Introduction of either the full-length clone R3-OFF or the partial cloneR3-OF in the forward orientation resulted in expression of type IIIreceptor. L6 cells transfected with the cDNA clones in the reverseorientation did not express this protein. Importantly, the apparent sizeof the protein core of the type III receptor in cells transformed withthe R3-OF clone is smaller than that from R3-OFF transformed cells,consistent with the difference in the sizes of the protein corespredicted from their nucleic acid sequences.

Surprisingly, binding of radio-labeled ligand to the type II receptorwas increased by 2.5 fold in cells expressing the type III cDNA. Bindingto the type I receptor was unchanged. This apparently specificup-regulation of ligand-binding to the type II receptor was evident inall of the 15 stably transfected L6 cell lines analyzed to date.Furthermore, this effect seems to be mediated equally well by thefull-length clone or a truncated clone (R3-OF) that lacks thecytoplasmic domain of TGF-β type III receptor was expressed.

Expression of type III receptor mRNA was assessed by Northern blotanalysis and RNA blot analysis. Northern gel analysis showed that thetype III receptor mRNA is expressed as a single 6 kb message in severalrat tissues. RNA dot blot analysis of several different tissue culturecell lines was also carried out. Cells of mouse origin (MEL and YH16)appear to express a smaller (˜5.5 kb) message for the type III mRNA thanthose of pig, rat and human origin. In all of these cells, expression orabsence of the type III mRNA is consistent with the expression orabsence of detectable cell surface type III receptors, with the notableexception of the retinoblastoma cell lines (Y79, Weri-1, Weri-24, andWeri-27). These cells lack detectable surface expression of type IIIreceptor, which confirms an earlier report. (Kimchi, A. et al., Science240:196-198 (1988)). It is striking that the type III receptor mRNA isexpressed in these cells at a level comparable to that of other cellsthat do indeed express type III receptor proteins at readily detectablelevels. It appears that TGF-β receptor III expression, which issubstantial in normal retinoblasts (AD12), has been down-regulated inthese retinoblastoma tumor cells, perhaps through post-transcriptionalmechanisms.

The nucleotide sequence full reading frame along with flanking sequencesof the full-length cDNA clone R3-OFF was determined and is presented inFIGS. 1A-1C. The reading frame encodes a protein of 853 amino acidresidues, which is compatible with the 100 kD size observed for thefully deglycosylated TGF-β1 type III receptor. The identity of thereceptor as TGF-β type III was verified by searching for segments of theputative transcription product which included the peptide sequencesdetermined by microsequencing of the isolated type III receptor. (SeeExample 1) As indicated in FIGS. 1A-1C, two segments of derived protein(underlined and italicized, residues 378-388 and 427-434) preciselymatch with the amino acid sequences of two peptides (I and III)determined from direct biochemical analysis of the purified type IIIreceptor.

Further analysis showed that TGF-β type III binding protein has anunusual structure for a cytokine receptor. Hydropathy analysis indicatesthat the protein includes a N-terminal signal sequence, followed by along, hydrophilic N-terminal region. A 27 residue region of stronghydrophobicity (underlined in FIGS. 1A-1C, residues 786-812) toward theC-terminus represents the single putative transmembrane domain. Thissuggests that nearly all of the receptor which is an N-terminalextracellular domain is anchored to the plasma membrane near itsC-terminus. A relatively small C-terminal tail of 41 residues representsthe cytoplasmic domain.

Analysis of related sequences provides few clues to function of TGF-βtype III protein. Only one other gene described to date, a glycoproteinexpressed in high quantities by endothelial cells and termed endoglin,contains a related amino acid sequence. The most homologous regionsbetween the sequences of the type III receptor and endoglin (74%) fallsprimarily in the putative transmembrane and cytoplasmic domains. Similarto the general structure of type III receptor, endoglin is aglycoprotein which contains a large hydrophilic N-terminal domain whichis presumably extracellular, followed by a putative transmembrane domainand a short cytoplasmic tail of 47 amino acid residues. The biologicalrole of endoglin is still unclear at present, although it has beensuggested that it may involved in cell-cell recognition throughinteractions of an “RGD” sequence on its ectodomain with other adhesionmolecules. Unlike the TGF-β type III receptor, endoglin does not carryGAG groups.

Isolation of TGF-β Type II Receptor

The cDNA encoding the type II TGF-β receptor was also isolated, usingexpression cloning in COS cells. A full-length cDNA (designated clone3FF) was isolated by high stringency hybridization from a human HepG2cell cDNA library. Analysis showed that the corresponding message is a 5kb message which is expressed in different cell lines and tissues.Sequence analysis indicated that the cDNA has an open reading frameencoding a core 567 amino acid residue protein. The nucleotide sequenceof the full-length type II TGF-β receptor cDNA clone 3FF is shown inFIGS. 2A-2B; the amino acid sequence is represented in FIG. 3.

The 567 amino acid residue protein has a single putative transmembranedomain, several consensus glycosylation sites, and a putativeintracellular serine/threonine kinase domain. The predicted size of theencoded protein core is ˜60 kd, which is too large for a type I TGF-βreceptor. Instead, crosslinking experiments using iodinated TGF-β andCOS cells transiently transfected with clone 3FF shows over-expressionof a protein approximately 70-80 kd which corresponds to the size oftype II TGF-β receptors. Thus, clone 3FF encodes a protein thatspecifically binds TGF-β and has an expressed protein size of 70-80 kd,both characteristic of the type II TGF-β receptor.

Uses of the Cloned TGF-β Receptors and Related Products

Plasmid 3FF was deposited in accordance with the provisions of theBudapest Treaty at the American Type Culture Collection (ATCC), 10801University Boulevard, Manassas, Va., 20110-2209, U.S.A. on Nov. 12,1997, and assigned Accession Number ATCC 209455.

For the first time, as a result of the work described herein, DNAsencoding two of the three high affinity cell-surface TGF-β receptorshave been isolated, their sequences and expression patterns determinedand the encoded proteins characterized. Expression of the TGF-β type IIIreceptor in cells which do not normally express the receptor, followedby ligand binding assay, verified that the cloned type IIIreceptor-encoding DNA (i.e., either the full-length clone R3-OFF or thepartial clone R3-OF) encoded the receptor. In addition, the workdescribed herein resulted in the surprising finding that binding ofTGF-β to type II receptors in cells expressing the type III DNA wasincreased by 2.5 fold.

Additional insight into the role of the TCF-β type III receptor and itsinteraction with TGF-β type II receptor is a result of the workdescribed. For example, the role of TGF-β type III receptor is unclear,but it has been proposed that it serves a most unusual function ofattracting and concentrating TGF-βs for eventual transfer to closelysituated signal-transducing receptors. While most cytokines bind to asingle cell surface receptor, members of the TGF-β family bind withgreater or lesser affinity to three distinct cell surface proteins. Thishas raised the question of why these three receptors are displayed bymost cell types and whether they subserve distinct functions. Evidenceobtained to date suggests that the type III receptor may performfunctions quite different from those of types I and II. Thus, type IIIis substantially modified by GAGs while types I and II appear to carryprimarily the N-linked (and perhaps O-linked) sidechains that arecharacteristic of most growth factor receptors. In addition, variantcells that have been selected for their ability to resist TGF-β-inducedgrowth inhibition show the absence of Type I or Type II receptors whilecontinuing to display Type III receptors. Together, these data havecaused some to propose that the Type I and II receptors represent bonafide signal-transducing receptors while the type III receptor, describedhere, plays another distinct role in the cell.

It remains possible that the type III receptor serves a most unusualfunction of attracting and concentrating TGF-βs on the cell surface foreventual transfer to closely situated signal-transducing receptors. Sucha function would be unprecedented for a proteinaceous receptor, althoughheparin sulfate has been shown to activate basic FGF by binding to thisgrowth factor prior to FGF association with its receptor (Yayon, A. etal., Cell 64:841-848 (1991)) Parenthetically, since the type IIIreceptor also contains large quantities of heparan sulfate side-chains,it may also bind and present basic FGF to its receptor.

Evidence that is consistent with the role for the type III receptorcomes from the work with L6 rat myoblast cells which is describedherein. As described above, in L6 cells overexpressing type IIIreceptor, the binding of radiolabelled TGF-β to the type II receptor isincreased several fold when compared with that seen with parental cells.Further assessment of TGF-β type III function and interaction with typeII and type I receptors will be needed to answer these questions and canbe carried out using the materials and methods described here.

TGF-β receptors, both type III and type II, can be identified in otherspecies, using all or a portion of the DNA encoding the receptor to beidentified as a probe and methods described herein. For example, all ora portion of the DNA sequence encoding TGF-β type III receptor (shown inFIGS. 1A-1C) or all or a portion of the DNA sequence encoding TGF-β typeII receptor (shown in FIGS. 2A-2B) can be used to identify equivalentsequences in other animals. Stringency conditions used can be varied, asneeded, to identify equivalent sequences in other species. Once aputative TGF-β receptor type III or type II-encoding sequence has beenidentified, whether it encodes the respective receptor type can bedetermined using known methods, such as described herein forverification that the cDNA insert of full-length clone R3-OFF and thecDNA insert of partial clone R3-OF encode the type III receptor. Forexample, DNA isolated in this manner can be expressed in an appropriatehost cell which does not express the receptor mRNA or the surfacereceptor (e.g., L6 rat skeleton muscle myoblasts) and analyzed by ligandbinding (TGF-β binding) assay, as described herein.

Also as a result of the work described herein, antibodies (polyclonal ormonoclonal) specific for the cloned TGF-β type III or the clones TGF-βtype II receptor can be produced, using known methods. Such antibodiesand host cells (e.g., hybridoma cells) producing the antibodies are alsothe subject of the present invention. Antibodies specific for the clonedTGF-β receptor can be used to identify host cells expressing isolatedDNA thought to encode a TGF-β receptor. In addition, antibodies can beused to block or inhibit TGF-β activity. For example, antibodiesspecific for the cloned TGF-β type III receptor can be used to blockbinding of TGF-β to the receptor. They can be administered to anindividual for whom reduction of TGF-β binding is desirable, such as insome fibrotic disease (e.g., of skin, kidney and lung).

DNA and RNA encoding TGF-β type III receptor and DNA and RNA encodingTGF-β type II receptor are now available. As used herein, the term DNAor RNA encoding the respective TGF-β receptor includes anyoligodeoxynucleotide or oligodeoxyribonucleotide sequence which, uponexpression, results in production of a TGF-β receptor having thefunctional characteristics of the TGF-β receptor. That is, the presentinvention includes DNA and RNA which, upon expression in an appropriatehost cell, produces a TGF-β type III receptor which has an affinity forTGF-β similar to that of the TGF-β type III receptor on naturallyoccurring cell surfaces (e.g., it shows comparable affinities for allTGF-β isotypes). Similarly, the present invention includes DNA and RNAwhich, upon expression in an appropriate host cell, produces a TGF-βtype II receptor which has an affinity for TGF-β similar to that ofTGF-β type II receptor on naturally occurring cell surfaces (e.g., ithas a distinctive affinity for each member of the TGF-β family ofligands similar to that of the naturally occurring TGF-β type IIreceptor). The DNA or RNA can be produced in an appropriate host cell orcan be produced synthetically (e.g., by an amplification technique suchas PCR) or chemically.

The present invention also includes the isolated TGF-β type III receptorencoded by the nucleotide sequence of full-length R3-OFF, the isolatedTGF-β type III receptor encoded by the nucleotide sequence of partialclone R3-OF, the isolated TGF-β type II receptor encoded by thenucleotide sequence of full-length clone 3FF and TGF-β type III and typeII receptors which bind TGF-β isotypes with substantially the sameaffinity, The isolated TGF-β type III and type II receptors can beproduced by recombinant techniques, as described herein, or can beisolated from sources in which they occur naturally or synthesizedchemically. As used herein, the terms cloned TGF-β type III and clonedTGF-β type II receptors include the respective receptors identified asdescribed herein, and TGF-β type III and type II receptors (e.g., fromother species) which exhibit substantially the same affinity for theTGF-β isotypes as the respective receptors.

As described previously, cells in which the cloned TGF-β type IIIreceptor is expressed bind TGF-β in essentially the same manner as docells on which the type III receptor occurs naturally. Further analysisof ligand interactions with the cloned TGF-β type III receptor, basedupon site-directed mutagenesis of both TGF-β and the receptor, can becarried out to identify residues important for binding. For example, DNAhaving the sequence of FIGS. 1A-1C can be altered by adding, deleting orsubstituting at least one nucleotide, in order to produce a modified DNAsequence which encodes a modified cloned TGF-β type III receptor. Thefunctional characteristics of the modified receptor (e.g., itsTGF-β-binding ability and association of the binding with effectsnormally resulting from binding) can be assessed, using the methodsdescribed herein. Modification of the cloned TGF-β type III receptor canbe carried out to produce, for example, a form of the TGF-β type IIIreceptor, referred to herein as soluble TGF-β receptor, which is notmembrane bound and retains the ability to bind the TGF-β isotypes withan affinity substantially the same as the naturally-occurring receptor.Such a TGF-β type III receptor could be produced, using known geneticengineering or synthetic techniques; it could include none of thetransmembrane region present in the naturally-occurring TGF-β type IIIreceptor or only a small portion of that region (i.e., small enough notto interfere with its soluble nature). For example, it can include aminoacids 1 through 785 of the TGF-β type III sequence of FIGS. 1A-1C or aportion of that sequence sufficient to retain TGF-β binding ability(e.g., amino acids 24-785, which does not include the signal peptidecleavage site present in the first 23 amino acids). A soluble TGF-β typeII receptor (e.g., one which does not include the transmembrane andcytoplasmic domains) can also be produced. For example, it can includeamino acids 1 through 166, inclusive, of FIG. 3 or a sufficient portionthereof to retain TGF-β binding ability substantially the same as thatof TGF-β type II receptor.

The TGF-β type III receptor and/or type II receptor can be used fortherapeutic purposes. As described above, the TGF-β family of proteinsmediate a wide variety of cellular activities, including regulation ofcell growth, regulation of cell differentiation and control of cellmetabolism. TGF-β may be essential to cell function and most cellssynthesize TGF-β and have TGF-β cell surface receptors. Depending oncell type and environment, the effects of TGF-β vary: proliferation canbe stimulated or inhibited, differentiation can be induced orinterrupted and cell functions can be stimulated or suppressed. TGF-β ispresent from embryonic stages through adult life and, thus, can affectthese key processes throughout life. The similarities of a particularTGF-β (e.g., TGF-β1) across species and from cell to cell areconsiderable. For example, the amino acid sequence of a particular TGF-βand the nucleotide sequence of the gene which encodes it regardless ofsource, are essentially identical across species. This further suggeststhat TGF-β has a critical role in essential processes.

Specifically, TGF-β has been shown to have anti-inflammatory and immunesuppression capabilities, to play an important role in bone formation(by increasing osteoblast activity), inhibit cancer cell proliferationin culture, and control proliferation of glandular cells of theprostate. As a result, it has potential therapeutic applications inaltering certain immune system responses (and possibly in modifyingimmune-mediated diseases); in treating systemic bone disease (e.g.,osteoporosis) and conditions in which bone growth is to be enhanced(e.g., repair of broken bones) and in controlling growth and metastasisof cancer cells. In addition, TGF-β appears to play a role indetermining whether some cell types undergo or do not undergo mitosis.In this respect, TGF-β may play an important role in tissue repair. Somediseases or conditions appear to involve low production or chronicoverproduction of TGF-β. (For example, results of animal studies suggestthat there is a correlation between the over production of TGF-β anddiseases characterized by fibrosis in the lung, kidney, liver or inviral mediated immune expression.)

Clearly, TGF-β has key roles in body processes and numerous relatedpotential clinical or therapeutic applications in wound healing, cancer,immune therapy and bone therapy. Availability of TGF-β receptor genes,the encoded products and methods of using them in vitro and in vivoprovides an additional ability to control or regulate TGF-β activity andeffect in the body. For example, the TGF-β type II or type III receptorencoded by the type II or the type III receptor genes of the subjectinvention can be used, as appropriate, to alter the effects of TGF-β(e.g., to enhance the effect of TGF-β in the body or to inhibit orreduce (totally or partially) its effects). It is also possible toadminister to an individual in whom TGF-β bound to TGF-β type IIIreceptor, such as soluble TGF-β type III receptor. The present inventionprovides both a TGF-β agonist and a TGF-β antagonist. For thesepurposes, DNA gene encoding the entire TGF-β type II or type IIIreceptor, the encoded type II or type III receptor or a soluble form ofeither receptor can be used. Alternatively, antibodies or other ligandsdesigned based upon these sequences or specific for them can be used forthis purpose.

Knowledge of the amino acid sequences of TGF-β type III and type IIreceptors makes it possible to better understand their structure and todesign compounds which interfere with binding of the receptor withTGF-β. It makes possible identification of existing compounds and designof new compounds which are type III and/or type II receptor antagonists.

Cells expressing the type III and/or type II receptors of the presentinvention can be used to screen compounds for their ability to interferewith (block totally or partially) TGF binding to the receptors. Forexample, cells which do not express TGF-β type III receptor (e.g., L6rat skeleton muscle myoblasts) but have been modified to do so byincorporation of the type III cDNA in an appropriate vector can be usedfor this purpose. A compound to be assessed is added, for example, totissue culture dishes containing type III expressing cells, along withlabeled TGF-β. As a control, the same concentration of labeled TGF-β isadded to tissue culture dishes containing the same type of cells. Aftersufficient time for binding of TGF-β to the receptor to occur, bindingof labeled TGF-β to the cells is assessed, using known methods (e.g., bymeans of a gamma counter) and the extent to which it occurred in thepresence and in the absence of the compound to be assessed isdetermined. Comparison of the two values show whether the test compoundblocked TGF-β binding to the receptor (i.e., less binding in thepresence of the compound than in its absence is evidence that the testcompound has blocked binding of TGF-β to the TGF-β type III receptor).

Alternatively, a cell line expressing the TGF-β receptor or cellsexpressing microinjected TGF-β receptor RNA, can be used to assesscompounds for their ability to block TGF-β binding to the receptor. Inthis embodiment, a compound to be assessed is added to tissue culturedishes containing the cell line cells expressing microinjected TGF-βreceptor RNA, along with TGF-β. As a control, TGF-β alone is added tothe same type of cells expressing microinjected endothelin receptor RNA.After sufficient time for binding of TGF-β to the receptor to occur, theextent to which binding occurred is measured, both in the presence andin the absence of the compound to be assessed. Comparison of the twovalues shows whether the compound blocked TGF-β binding to the receptor.The TGF-β type III and type II receptors can also be used to identifyTGF-β-like substances, to purify TGF-β and to identify TGF-β regionswhich are responsible for binding to the respective receptors. Forexample, the type III receptor can be used in an affinity-based methodto identify substances which bind the receptor in a manner similar toTGF-β.

The invention will now be illustrated by the following examples, whichare not intended to be limiting in any way.

EXAMPLES

Materials and methods used in Examples 1-5 are described below.

Materials

The following is a description of materials used in the work describedherein.

Recombinant human TGF-β1 was provided by Rik Derynck of Genentech.COS-M6 cells were provided by Brian Seed of the Massachusetts GeneralHospital and Alejandro Aruffo of Bristol-Myers-Squibb. Heparitinase wasprovided by Tetsuhito Kojima and Robert Rosenberg of MIT. LLC-PK₁ cellswere a gift of Dennis Ausiello of the Massachusetts General Hospital.YH-16 cell were a gift of Edward Yeh of the Massachusetts GeneralHospital. 3-4 cells were a gift of Eugene Kaji of the WhiteheadInstitute for Biomedical Research. All other cell lines were purchasedfrom ATCC and grown as specified by the supplier, except as noted.

Expression Cloning

Construction of cDNA Library and Generation of Plasmid Pools

10 μg polyadenylated mRNA was prepared from A10 cells by theproteinase-K/SDS method (Gonda et al., Molec. Cell. Biol. 2:617-624(1982)). Double stranded cDNA was synthesized and linkered tononpalindromic BstX1 adaptors as described by Seed, B. and A. Aruffo,Proc. Natl. Acad. Sci. USA 84:3365-3369 (1987). Acaptored cDNA wassize-fractionated on a 5 to 20% potassium acetate gradient, and insertsgreater than 1 kb were ligated to the plasmid vector pcDNA-1, andelectroporated in the E. coli MC1061/P3, yielding a primary library witha titer of >10⁷ recombinants. A portion of the cDNA was plated as poolsof ˜1×10⁴ recombinant bacteria colonies grown on 15 cm petri dishes withLuria-broth agar containing 7.5 mg/ml tetracycline and 12.5 mg/mlampicillin. Cells were scraped off the plates in 10 mls of Luria-broth,and glycerol stocks of pooled bacteria were stored at −70° C. Theremaining bacteria was used to purify plasmid DNA using the alkalinelysis mini-prep method (Sambrook, J. et al., Molecular Cloning: ALaboratory Manual, 2d Ed. (Cold Spring Harbor, N.Y., Cold Spring HarborLaboratory Press (1989)).

COS Cell Transfections and Binding Assay

Plasmid pools (each representing ˜1×10⁴ clones) were transfected intoCOS-M6 (subdlone of COS-7 cells) by the DEAE-dextran/chloroquine methoddescribed by Seed, B. and A. Aruffo, Proc. Natl. Acad. Sci. USA84:3365-3369 (1987). Forty-eight hours after transfection, cells wereincubated with 50 pM¹²⁵I-TGF-β1 (100 to 200 Ci/mmol) for 4 hours at 4°C.), autoradiographic analysis of transfected cells was performed usingNT-B2 photographic emulsion (Kodak) essentially as described (Gearing,D. P. et al., EMBO J. 8:3667-3676 (1989)). After development of slides,cells were air-dried and mounted with mounting media and a glasscoverslip. Slides were analyzed under an Olympus OM-T1 invertedphase-contrast microscope using a dissection trans-illuminator fordarkfield illumination.

Subdivision of Positive Pool

Of 86 pools screened, one pool (#13) was identified as positive and aglycerol stock of bacteria corresponding to this pool was titered and 25pools of 1000 clones were generated and miniprep plasmid from thesepools were transfected into COS cells as described above. Severalpositive pools of 1000 were identified, and one was replated as 25plates of 100 colonies. A replica was made of this positive plate andharvested. Once a positive pool was identified, individual colonies werepicked from the corresponding master plate and grown overnight in 3 mlliquid culture. A 2-dimensional grid representing the 100 clones wasgenerated and a single clone, R3-OF, was isolated.

Cloning of R3-OFF

A 208F rat fibroblast library in lambda ZAP II (Stratagene) was screenedat high stringency with clone R3-OF insert, and several clones with ˜6kb inserts were isolated, one of which is referred to as R3-OFF.

DNA Sequencing and Seguence Analysis

Double-stranded DNA was sequenced by the dideoxy chain terminationmethod using Sequenase reagents (United States Biochemicals). Comparisonof the sequence to the data bases was performed using BLAST (Altschcul,S. F. et al., J. Mol. Biol. 215:403-410 (1990)).

Iodination of TGF-β

TGF-β1 was iodinated using the chloramine-T method as described(Cheifetz, S. and J. L. Andres, J. Biol. Chem. 263:16984-16991 (1988)).

Chemical Cross-Linking

Transfected COS cells grown on 10 cm dishes or subconfluent L6 and A-10cells grown on 3.5 cm dishes were incubated with ¹²⁵I-TGF-β1 in bindingbuffer (Frebs-Ringer buffered with 20 mM Hepes, pH 7.5, 5 mM MgSO₄, 0.5%BSA), washed 4 times with ice-cold binding buffer without BSA, andincubated for 15 minutes with binding buffer without BSA containing 60ng/ml disuccinimidyl suberate at 4° C. under constant rotation.Crosslinking was terminated by addition of 7% sucrose in binding buffer.Cells were scraped, collected and pelleted by centrifugation, thenresuspended in lysis buffer (10 mM Tris, pH 7.4, 1 mM EDTA, pH 8.0, 1%Triton-X 100, 10 μg/ml of pepstatin, 10 μg/ml leupeptin, 10 μg/mlantipain, 100 μg/m; benzamidine hydrochloride, 100 μg/ml soybean trypsininhibitor, 50 μg/ml aprotonin, and 1 mM phenylmethylsulfonyl fluoride).Solubilized material was analyzed by 7% SDS-PAGE and subjected toautoradiographic analysis by exposure to X-AR film (Kodak) at −70° C.

Enzymatic Digestion

Digestion of solubilized TGF-b receptors with chondroitinase andheparitinase was performed as described (Cheifetz, S. and J. L. Andres,J. Biol. Chem. 263:16984-16991 (1988); Segarini, P. R. and S. M.Seyedin, J. Biol. Chem., 263: 8366-8370 (1988).

Generation of Stable Cell Lines

L6 myoblasts were split 1:10 into 10 cm dishes and transfected thefollowing day by the calcium phosphate method (Chen, C. and H. Okayama,Molec. Cell. Biol. 7:2745-2752 (1987)) with clones R3-OF or R3-OFF inthe forward and reverse orientations in the vector pcDNA-neo(InVitrogen). Cells were subjected to selection in the presence of G418(Geneticin, GIBCO) for several weeks until individual colonies werevisible by the naked eye. These clones were isolated and amplified.

RNA Blot Analyses

Rat tissue polyadenylated mRNA was prepared by the lithium chloride/ureamethod (Auffrey, C. and F. Raugeon, Eur. J. Biochemistry 107:303-313(1980), followed by oligo-dT cellulose chromatography (Aviv and Leder,1972). Polyadenylated mRNA from cell lines was prepared by theproteinase K/SDS method (Gonda, T. J. et al., Molec. Cell. Biol.2:617-624 (1982)). Samples of mRNA were resolved by electrophoresis on1% agarose-2.2M formaldehyde gels, blotted onto nylon membranes(Biotrns, ICN) and incubated with the 2.9 kb insert of clone Re-OFlabeled with ³²P by random priming as probe (Sambrook, J. et al.,Molecular Cloning: A Laboratory Manual, 2d Ed., Cold Spring Harbor,N.Y., Cold Spring Harbor Laboratory Press, (1989). Hybridizations wereperformed at 42° C. in hybridization buffer containing 50% formamideovernight, and blots were washed at 55° C. in 0.2×SSC, 0.1% SDS, beforeexposure to X-AR film at −70° C.

Example 1

Production of Anti-Type III Receptor Protein Antibodies andMicrosequencing and Microsequencing of Peptides Resulting from PartialProteolysis of Purified Type III Receptor

Initially cellular proteoglycans were purified from human placenta andthen subjected to enzymatic deglycosylation with heparitinase andchondroitinase. Protein cores in the molecular weight range of 100-130kilodaltons were further purified by preparative gel electrophoresis;these should include the type III receptor. This partially purifiedmaterial was used as an immunogen in mice. After screening 850 hybridomalines developed from immunized mice, three lines were found to produceantibodies that specifically recognized and immuno-precipitated adeglycosylated polypeptide species of 100-120 kD. This species could beradiolabelled by incubation of whole cells with ¹²⁵I-TGF-β followed bycovalent cross-linking. Its size is consistent with that of the proteincore previously reported for the type III receptor. (Massague, J., Annu.Rev. Cell. Biol. 6:597-641 (1990))

Monoclonal antibody 94 was used to purify the type III receptor from ratliver by affinity-chromatography. The purified receptor was subjected topartial proteolysis and the resulting peptides were resolved by highpressure liquid chromatography. Several peptides were subjected tomicrosequencing and yielded the following oligopeptide sequences:

Peptide I: ILLDPDHPPAL (SEQ ID NO:1) Peptide II: QAPFPINFMIA (SEQ IDNO:2)

Peptide III: QPIVPSVQ (SEQ ID NO:3) Peptide IV: FYVEQGYGR (SEQ ID NO:4)

These peptide sequences were used to construct degenerateoligonucleotides that served either as primers in a cloning strategyusing the polymerase chain reaction (PCR) or as probes in screening cDNAlibraries. While this strategy was not productive, the oligopeptidesequences proved useful in verifying the receptor clones isolated by thesecond, alternative strategy (see Example 2).

Example 2

Expression Cloning of the Type III Receptor cDNA

An expression cloning strategy in COS cells, a procedure which takesadvantage of the considerable amplification of individual cDNAs intransfected COS cells was used as an alternative means to isolate TGF-βreceptor clones. Such amplification is mediated by SV40 large T antigenexpressed by the COS cells interacting with a SV40 origin of replicationin the cDNA vector. Gearing, D. et al., EMBO J. 8:3667-3676 (1989); Lin,H. Y., et al., Proc. Natl. Acad. Sci. 88:3185-3189 (1991a); Lin, H. Y.et al., Science, in press (1991); Mathews, L. S. and Vale, W. W., Cell65:973-982 (1991).

The strategy involved the construction of a cDNA library from A-10cells, a rat vascular smooth muscle cell line that expresses all threehigh-affinity TGF-β receptors. The resulting cDNAs were inserted intothe vector pcDNA-1, which carries the CMV transcriptional promoter andthe SV40 origin of replication. The resulting library was then dividedinto pools of 10,000 independent recombinants each and DNA from eachpool was transfected into 1.5×10⁶ COS-7 cells grown on glass flaskettesby means of DEAE-dextran transfection procedure. Aruffo, A. and Seed,B., Proc. Natl. Acad. Sci., U.S.A. 84:8573-8577 (1987); Gearing, D. etal., EMBO J. 8:3667-3676 (1989); Mathews, L. S. and Vale, W. W., Cell65:973-982 (1991). The transfected cells were cultured for 48-60 hoursand then exposed to radiolabelled TGF-β1 for four hours. Following thistreatment, the glass slides carrying these cells were washed extensivelyand fixed. These slides were dipped in liquid photographic emulsion andexamined by darkfield microscopy. While all of the receptor genes clonedto date by this procedure have undetectable or low constitutive levelsof expression in COS cells, we were hindered by the fact thatuntransfected COS cells already express substantial amounts of type IIITGF-β receptor. Such expression, estimated to be approximately 2×10⁵receptor molecules per cell, might well have generated an unacceptablyhigh level of background binding. However, since the detection procedureinvolves visualizing radiolabelled ligand-binding on individual cells,it was hoped that identifying occasional cells expressing substantiallyhigher levels of vector-encoded receptor would be possible. This hopewas vindicated in the initial experiments.

After screening nearly one million cDNA clones in this manner, a glassslide containing 20 positive transfectants was identified. The originalpool of expression constructs from which one such transfectant wasderived was split into 25 subpools of 1000 clones each and these weresubjected to a second round of screening. Two further rounds ofsib-selection resulted in the isolation of a cDNA clone (R3-OF) with a2.9 kb insert that induced high levels of TGF-β-binding proteins inapproximately 10% of COS cells into which it was transfected.

The specificity of this binding was validated by showing that additionof a 200-fold excess of unlabeled TGF-β competitor strongly reducedbinding of ¹²⁵I-TGF-β to transfected cells. By taking into account atransfection efficiency of 10% and the high background of endogenousreceptor expression, we calculated that the level of total ¹²⁵I-TGF-βbinding to each glass slide of cells transfected with this cDNA clonewas only 2-fold above the level seen with mock transfectants (data notshown). Nonetheless, this marginal increase in ligand-binding wassufficient to identify rare transfectants amidst a large field of cellsexpressing this background level of receptor.

The R3-OF cDNA encoded an open reading frame of 836 amino acid residuesof which the 3′ most 18 were encoded by vector sequence, clearlyindicating that clone R3-OF was an incomplete cDNA insert which endedprematurely at the codon specifying alanine 818 (FIG. 3). R3-OF was usedas a probe to isolate a full-length cDNA from a rat 208F lambda phagelibrary. This clone, termed R3-OFF, was 6 kb in length and encoded aprotein of 853 amino acids; its sequence was co-linear with that ofclone R3-OF.

Example 3

Characterization of the Product of the Full Length Clone R3-OFF

Characterization of the product of the full length clone R3-OFF wasundertaken in order to determine which of the three TGF-β receptors itspecified. To do so, COS transfectants were incubated withradioiodinated TGF-β, chemical crosslinker was added and the labelledreceptors were resolved by polyacrylamide gel electrophoresis.

Labelling of cell surface TGF-β receptors in this way resulted in thedetection of three distinct species on the surface of COS cells, asextensively by others (Massague, J. et al., Ann. NY Acad. Sci. 593:59-72(1990). These included the two lower molecular weight type I and IIreceptors (65 and 85 kD) and the higher molecular weight type IIIproteoglycan, which migrated as a diffuse band of 280-330 kd. Enzymatictreatment of the proteoglycan with chondroitinase and heparitinaseyielded a core protein of approximately 100 kd. Binding to all threereceptor types was specific, in that it was completed by 200-fold excessof unlabeled TGF-β1.

Transfecting the R3-OFF cDNA caused a two-fold increase in expression ofthe type III receptor. When a cell lysate derived from COS cellstransfected with clone R3-OFF was treated with deglycosylating enzymes,the heterogenous 280-330 kd band was converted to a protein core whichco-migrated with the type III protein core seen in untransfetted A10cells. Importantly, the recombinant protein core migrates differentlyfrom the endogenous COS cell type III protein core.

These observations were confirmed and extended in experiments usingstably transfected cells expressing the R3-OFF cDNA. L6 rat skeletonmuscle myoblasts normally do not express detectable type III mRNA orendogenous type III receptor (determined by radiolabelled ligand-bindingassay). Such cells were transfected with the isolated cDNA in the vectorpcDNA-neo. Cell clones stably expressing this clone in both the forwardand reverse orientations with respect to the CMV promoter were isolatedand analyzed by ligand-binding assay.

Introduction of either the full length clone R3-OFF or the partial cloneR3-OF in the forward orientation led to the de novo expression of thetype III receptor. L6 cells transfected with the cDNA in reversedorientation did not express this protein. The apparent size of theprotein core of the type III receptor in cells transfected with theR3-OF clone is smaller than that expressed by R3-OFF transfected cells,consistent with the difference in the sizes of the protein corespredicted from the respective nucleic acid sequences (FIGS. 1A-1C).

Unexpectedly the amount of radio-labelled ligand cross-linked to thetype II receptor is increased by 2.5 fold in cells expressing the typeIII cDNA, while the amount cross-linked to the type I receptor remainedunchanged. This apparent specific up-regulation of ligand-binding to thetype II receptor could be detected with all of the 15 stably transfectedL6 cell lines analyzed so far. This effect seems to be mediated by thetruncated clone R3-OF which lacks the cytoplasmic domain as well as bythe full-length clone R3-OFF.

Example 4

Expression of Type III Receptor

Northern blot analysis demonstrated that the type III receptor mRNA isexpressed as a single 6 kb message in several rat tissues. The level ofmRNA expression in the liver was less than in other tissues, a resultexpected from earlier surveys of various tissues using radioiodinatedTGF-β1. Based on this information, it appears that clone R3-OFF, with a˜6 kb cDNA insert, represents a full length rat type III cDNA clone.

Cells of mouse origin (MEL and YH16) appear to express a smaller (˜5.5kb) message for the type III mRNA than those of pig, rat and humanorigin. In all of these cells, expression or absence of the type IIImRNA is consistent with the expression or absence of detectable cellsurface type III receptors with the notable exception of theretinoblastoma cell lines (Y79, Weri-1, Weri-24, and Weri-27). Thesecells have previously been shown to lack detectable surface expressionof type III receptor, a result confirmed by our own unpublished work. Itis striking that the type III receptor mRNA is expressed in these cellsat a level comparable to that of other cells that do indeed express typeIII receptor proteins at readily detectable levels. At this moment, wecan only conclude that TGF-β receptor III expression, which issubstantial in normal retinoblasts (AD12), has been down-regulated inthese retinoblastoma tumor cells, perhaps through post-transcriptionalmechanisms.

Example 5

Sequence Analysis of the Full-Length Type III cDNA

The full-length cDNA clone (R3-OFF), described in Example 4, wassubjected to sequence analysis. The full reading frame along withflanking sequences is presented in FIGS. 1A-1C. This reading frameencodes a protein of 853 amino acid residues, which is compatible withthe 100 kD observed for the fully deglycosylated TGF-β type IIIreceptor.

Two segments of derived protein sequence (underlined and italicized,residues 378-388 and 427-434) precisely match those determined earlierfrom direct biochemical analysis of the purified receptor protein. Thisfurther secured the identity of this isolated cDNA clone as encoding therat type III receptor.

This TGF-β binding protein has an unusual structure for a cytokinereceptor. Hydropathy analysis indicates a N-terminal signal sequence,followed by a long, hydrophilic N-terminal region (Kyte, J. and R. F.Doolittle, J. Mol. Biol. 157:105-132 (1982)). A 27 residue region ofstrong hydrophobicity (underlined, residues 786-812) toward theC-terminus represents the single putative transmembrane domain. Thissuggests that nearly all of the receptor is composed of an N-terminalextracellular domain that is anchored to the plasma membrane near itsC-terminus. A relatively short C-terminal tail of 41 residues representsthe putative cytoplasmic domain.

The clone R3-OF was also analyzed and found to be a truncated version ofR3-OFF, with an identical open reading frame but whose last encodedresidue is alanine 818 (FIGS. 1A-1C).

In R3-OFF there are six consensus N-linked glycosylation sites and 15cysteines (indicated in FIGS. 1A-1C). There is at least one consensusglycosaminoglycan addition site at serine 535 (Bernfield, M. and K. C.Hooper, Ann. N.Y. Acad. Sci. in press (1991), and numerous Ser-Glyresidues that are potential sites for GAG conjugation. A consensusprotein kinase C site is also present at residue 817.

Only one other gene described to date, a glycoprotein expressed in highquantities by endothelial cells and termed endoglin (Gougos and Letarte,1990), contains a related amino acid sequence. Overall, there is ˜30%identity with the type III receptor over the entire 645 amino acidresidue length of endoglin. The most homologous regions between thesequences of the type III receptor and endoglin (74% identity) fallsprimarily in the putative transmembrane and cytoplasmic domains. Similarto the general structure of type III receptor, endoglin is aglycoprotein which contains a large hydrophilic and presumablyextracellular N-terminal domain followed by a putative transmembranedomain and a short cytoplasmic tail of 47 amino acid residues. Thebiological role of endoglin is unclear, though it has been suggestedthat it may involve cell-cell recognition through interactions of an“RGD” sequence on its ectodomain with other adhesion molecules. Unlikethe TGF-β type III receptor, endoglin does not carry GAG groups.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingnot more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

8 11 amino acids amino acid double linear unknown 1 Ile Leu Leu Asp ProAsp His Pro Pro Ala Leu 1 5 10 11 amino acids amino acid double linearunknown 2 Gln Ala Pro Phe Pro Ile Asn Phe Met Ile Ala 1 5 10 8 aminoacids amino acid double linear unknown 3 Gln Pro Ile Val Pro Ser Val Gln1 5 9 amino acids amino acid double linear unknown 4 Phe Tyr Val Glu GlnGly Tyr Gly Arg 1 5 3237 base pairs nucleic acid double linear DNA(genomic) unknown CDS 241..2799 5 CAGGAGGTGA AAGTCCCCGG CGGTCCGGATGGCGCAGTTG CACTGCGCTG CTGAGCTCGC 60 GGCCGCCTGC GCACACTGGG GGGACTCGCTTCGGCTAGTA ACTCCTCCAC CTCGCGGCGG 120 ACGACCGGTC CTGGACACGC TGCCTGCGAGGCAAGTTGAA CAGTGCAGAG AAGGATCTTA 180 AAGCTACACC CGACTTGCCA CGATTGCCTTCAATCTGAAG AACCAAAGGC TGTTGGAGAG 240 ATG GCA GTG ACA TCC CAC CAC ATG ATCCCG GTG ATG GTT GTC CTG ATG 288 Met Ala Val Thr Ser His His Met Ile ProVal Met Val Val Leu Met 1 5 10 15 AGC GCC TGC CTG GCC ACC GCC GGT CCAGAG CCC AGC ACC CGG TGT GAA 336 Ser Ala Cys Leu Ala Thr Ala Gly Pro GluPro Ser Thr Arg Cys Glu 20 25 30 CTG TCA CCA ATC AAC GCC TCT CAC CCA GTCCAG GCC TTG ATG GAG AGC 384 Leu Ser Pro Ile Asn Ala Ser His Pro Val GlnAla Leu Met Glu Ser 35 40 45 TTC ACC GTT CTG TCT GGC TGT GCC AGC AGA GGCACC ACC GGG CTG CCA 432 Phe Thr Val Leu Ser Gly Cys Ala Ser Arg Gly ThrThr Gly Leu Pro 50 55 60 AGG GAG GTC CAT GTC CTA AAC CTC CGA AGT ACA GATCAG GGA CCA GGC 480 Arg Glu Val His Val Leu Asn Leu Arg Ser Thr Asp GlnGly Pro Gly 65 70 75 80 CAG CGG CAG AGA GAG GTT ACC CTG CAC CTG AAC CCCATT GCC TCG GTG 528 Gln Arg Gln Arg Glu Val Thr Leu His Leu Asn Pro IleAla Ser Val 85 90 95 CAC ACT CAC CAC AAA CCT ATC GTG TTC CTG CTC AAC TCCCCC CAG CCC 576 His Thr His His Lys Pro Ile Val Phe Leu Leu Asn Ser ProGln Pro 100 105 110 CTG GTG TGG CAT CTG AAG ACG GAG AGA CTG GCC GCT GGTGTC CCC AGA 624 Leu Val Trp His Leu Lys Thr Glu Arg Leu Ala Ala Gly ValPro Arg 115 120 125 CTC TTC CTG GTT TCG GAG GGT TCT GTG GTC CAG TTT CCATCA GGA AAC 672 Leu Phe Leu Val Ser Glu Gly Ser Val Val Gln Phe Pro SerGly Asn 130 135 140 TTC TCC TTG ACA GCA GAA ACA GAG GAA AGG AAT TTC CCTCAA GAA AAT 720 Phe Ser Leu Thr Ala Glu Thr Glu Glu Arg Asn Phe Pro GlnGlu Asn 145 150 155 160 GAA CAT CTC GTG CGC TGG GCC CAA AAG GAA TAT GGAGCA GTG ACT TCG 768 Glu His Leu Val Arg Trp Ala Gln Lys Glu Tyr Gly AlaVal Thr Ser 165 170 175 TTC ACT GAA CTC AAG ATA GCA AGA AAC ATC TAT ATTAAA GTG GGA GAA 816 Phe Thr Glu Leu Lys Ile Ala Arg Asn Ile Tyr Ile LysVal Gly Glu 180 185 190 GAT CAA GTG TTT CCT CCT ACG TGT AAC ATA GGG AAGAAT TTC CTC TCA 864 Asp Gln Val Phe Pro Pro Thr Cys Asn Ile Gly Lys AsnPhe Leu Ser 195 200 205 CTC AAT TAC CTT GCC GAG TAC CTT CAA CCC AAA GCCGCC GAA GGT TGT 912 Leu Asn Tyr Leu Ala Glu Tyr Leu Gln Pro Lys Ala AlaGlu Gly Cys 210 215 220 GTC CTG CCC AGT CAG CCC CAT GAA AAG GAA GTA CACATC ATC GAG TTA 960 Val Leu Pro Ser Gln Pro His Glu Lys Glu Val His IleIle Glu Leu 225 230 235 240 ATT ACC CCC AGC TCG AAC CCT TAC AGC GCT TTCCAG GTG GAT ATA ATA 1008 Ile Thr Pro Ser Ser Asn Pro Tyr Ser Ala Phe GlnVal Asp Ile Ile 245 250 255 GTT GAC ATA CGA CCT GCT CAA GAG GAT CCC GAGGTG GTC AAA AAC CTT 1056 Val Asp Ile Arg Pro Ala Gln Glu Asp Pro Glu ValVal Lys Asn Leu 260 265 270 GTC CTG ATC TTG AAG TGC AAA AAG TCT GTC AACTGG GTG ATC AAG TCT 1104 Val Leu Ile Leu Lys Cys Lys Lys Ser Val Asn TrpVal Ile Lys Ser 275 280 285 TTT GAC GTC AAG GGA AAC TTG AAA GTC ATT GCTCCC AAC AGT ATC GGC 1152 Phe Asp Val Lys Gly Asn Leu Lys Val Ile Ala ProAsn Ser Ile Gly 290 295 300 TTT GGA AAA GAG AGT GAA CGA TCC ATG ACA ATGACC AAA TTG GTA AGA 1200 Phe Gly Lys Glu Ser Glu Arg Ser Met Thr Met ThrLys Leu Val Arg 305 310 315 320 GAT GAC ATC CCT TCC ACC CAA GAG AAT CTGATG AAG TGG GCA CTG GAC 1248 Asp Asp Ile Pro Ser Thr Gln Glu Asn Leu MetLys Trp Ala Leu Asp 325 330 335 AAT GGC TAC AGG CCA GTG ACG TCA TAC ACAATG GCT CCC GTG GCT AAT 1296 Asn Gly Tyr Arg Pro Val Thr Ser Tyr Thr MetAla Pro Val Ala Asn 340 345 350 AGA TTT CAT CTT CGG CTT GAG AAC AAC GAGGAG ATG AGA GAT GAG GAA 1344 Arg Phe His Leu Arg Leu Glu Asn Asn Glu GluMet Arg Asp Glu Glu 355 360 365 GTC CAC ACC ATT CCT CCT GAG CTT CGT ATCCTG CTG GAC CCT GAC CAC 1392 Val His Thr Ile Pro Pro Glu Leu Arg Ile LeuLeu Asp Pro Asp His 370 375 380 CCG CCC GCC CTG GAC AAC CCA CTC TTC CCAGGA GAG GGA AGC CCA AAT 1440 Pro Pro Ala Leu Asp Asn Pro Leu Phe Pro GlyGlu Gly Ser Pro Asn 385 390 395 400 GGT GGT CTC CCC TTT CCA TTC CCG GATATC CCC AGG AGA GGC TGG AAG 1488 Gly Gly Leu Pro Phe Pro Phe Pro Asp IlePro Arg Arg Gly Trp Lys 405 410 415 GAG GGC GAA GAT AGG ATC CCC CGG CCAAAG CAG CCC ATC GTT CCC AGT 1536 Glu Gly Glu Asp Arg Ile Pro Arg Pro LysGln Pro Ile Val Pro Ser 420 425 430 GTT CAA CTG CTT CCT GAC CAC CGA GAACCA GAA GAA GTG CAA GGG GGC 1584 Val Gln Leu Leu Pro Asp His Arg Glu ProGlu Glu Val Gln Gly Gly 435 440 445 GTG GAC ATC GCC CTG TCA GTC AAA TGTGAC CAT GAA AAG ATG GTC GTG 1632 Val Asp Ile Ala Leu Ser Val Lys Cys AspHis Glu Lys Met Val Val 450 455 460 GCT GTA GAC AAA GAC TCT TTC CAG ACCAAT GGC TAC TCA GGG ATG GAG 1680 Ala Val Asp Lys Asp Ser Phe Gln Thr AsnGly Tyr Ser Gly Met Glu 465 470 475 480 CTC ACC CTG TTG GAT CCT TCG TGTAAA GCC AAA ATG AAT GGT ACT CAC 1728 Leu Thr Leu Leu Asp Pro Ser Cys LysAla Lys Met Asn Gly Thr His 485 490 495 TTT GTT CTC GAG TCT CCC CTG AATGGC TGT GGT ACT CGA CAT CGG AGG 1776 Phe Val Leu Glu Ser Pro Leu Asn GlyCys Gly Thr Arg His Arg Arg 500 505 510 TCG ACC CCG GAT GGT GTG GTT TACTAT AAC TCT ATT GTG GTG CAG GCT 1824 Ser Thr Pro Asp Gly Val Val Tyr TyrAsn Ser Ile Val Val Gln Ala 515 520 525 CCG TCC CCT GGG GAT AGC AGT GGCTGG CCT GAT GGC TAT GAA GAC TTG 1872 Pro Ser Pro Gly Asp Ser Ser Gly TrpPro Asp Gly Tyr Glu Asp Leu 530 535 540 GAG TCA GGC GAT AAT GGA TTT CCTGGA GAC GGG GAT GAA GGA GAA ACT 1920 Glu Ser Gly Asp Asn Gly Phe Pro GlyAsp Gly Asp Glu Gly Glu Thr 545 550 555 560 GCC CCC CTG AGC CGA GCT GGAGTG GTG GTG TTT AAC TGC AGC TTG CGG 1968 Ala Pro Leu Ser Arg Ala Gly ValVal Val Phe Asn Cys Ser Leu Arg 565 570 575 CAG CTG AGG AAT CCC AGT GGCTTC CAG GGC CAG CTC GAT GGA AAT GCT 2016 Gln Leu Arg Asn Pro Ser Gly PheGln Gly Gln Leu Asp Gly Asn Ala 580 585 590 ACC TTC AAC ATG GAG CTG TATAAC ACA GAC CTC TTT CTG GTG CCC TCC 2064 Thr Phe Asn Met Glu Leu Tyr AsnThr Asp Leu Phe Leu Val Pro Ser 595 600 605 CCA GGG GTC TTC TCT GTG GCAGAG AAC GAG CAT GTT TAT GTT GAG GTG 2112 Pro Gly Val Phe Ser Val Ala GluAsn Glu His Val Tyr Val Glu Val 610 615 620 TCT GTC ACC AAG GCT GAC CAAGAT CTG GGA TTC GCC ATC CAA ACC TGC 2160 Ser Val Thr Lys Ala Asp Gln AspLeu Gly Phe Ala Ile Gln Thr Cys 625 630 635 640 TTT CTC TCT CCA TAC TCCAAC CCA GAC AGA ATG TCT GAT TAC ACC ATC 2208 Phe Leu Ser Pro Tyr Ser AsnPro Asp Arg Met Ser Asp Tyr Thr Ile 645 650 655 ATC GAG AAC ATC TGT CCGAAA GAC GAC TCT GTG AAG TTC TAC AGC TCC 2256 Ile Glu Asn Ile Cys Pro LysAsp Asp Ser Val Lys Phe Tyr Ser Ser 660 665 670 AAG AGA GTG CAC TTT CCCATC CCG CAT GCT GAG GTG GAC AAG AAG CGC 2304 Lys Arg Val His Phe Pro IlePro His Ala Glu Val Asp Lys Lys Arg 675 680 685 TTC AGC TTC CTG TTC AAGTCT GTG TTC AAC ACC TCC CTG CTC TTC CTG 2352 Phe Ser Phe Leu Phe Lys SerVal Phe Asn Thr Ser Leu Leu Phe Leu 690 695 700 CAC TGC GAG TTG ACT CTGTGC TCC AGG AAG AAG GGC TCC CTG AAG CTG 2400 His Cys Glu Leu Thr Leu CysSer Arg Lys Lys Gly Ser Leu Lys Leu 705 710 715 720 CCG AGG TGT GTG ACTCCT GAC GAC GCC TGC ACT TCT CTC GAT GCC ACC 2448 Pro Arg Cys Val Thr ProAsp Asp Ala Cys Thr Ser Leu Asp Ala Thr 725 730 735 ATG ATC TGG ACC ATGATG CAG AAT AAG AAG ACA TTC ACC AAG CCC CTG 2496 Met Ile Trp Thr Met MetGln Asn Lys Lys Thr Phe Thr Lys Pro Leu 740 745 750 GCT GTG GTC CTC CAGGTA GAC TAT AAA GAA AAT GTT CCC AGC ACT AAG 2544 Ala Val Val Leu Gln ValAsp Tyr Lys Glu Asn Val Pro Ser Thr Lys 755 760 765 GAT TCC AGT CCA ATTCCT CCT CCT CCT CCA CAG ATT TTC CAT GGC CTG 2592 Asp Ser Ser Pro Ile ProPro Pro Pro Pro Gln Ile Phe His Gly Leu 770 775 780 GAC ACG CTC ACC GTGATG GGC ATT GCA TTT GCA GCA TTT GTG ATC GGA 2640 Asp Thr Leu Thr Val MetGly Ile Ala Phe Ala Ala Phe Val Ile Gly 785 790 795 800 GCG CTC CTG ACGGGG GCC TTG TGG TAC ATC TAC TCC CAC ACA GGG GAG 2688 Ala Leu Leu Thr GlyAla Leu Trp Tyr Ile Tyr Ser His Thr Gly Glu 805 810 815 ACA GCA CGA AGGCAG CAA GTC CCT ACC TCG CCG CCA GCC TCG GAG AAC 2736 Thr Ala Arg Arg GlnGln Val Pro Thr Ser Pro Pro Ala Ser Glu Asn 820 825 830 AGC AGC GCG GCCCAC AGC ATC GGC AGC ACT CAG AGT ACC CCC TGC TCT 2784 Ser Ser Ala Ala HisSer Ile Gly Ser Thr Gln Ser Thr Pro Cys Ser 835 840 845 AGC AGC AGC ACAGCC TAGGTGGACA GACAGACGCC CGCCCACCGC AGCCAGGGCA 2839 Ser Ser Ser Thr Ala850 GGGCCCGATG CCAGTGCTGC GTGTCCACAG TCAGAAGTCT TGATCTGGGC TCCCTGTAAA2899 GAAAGAGTGA ATTTCAGTAT ACAGACAGCC AGTTCTACCC ACCCCTTACC ACGGCCCACA2959 TAAATGTGAC CCTGGGCATC TGTCACACGA AAGCTAAGCT GGTGGCCTTC CCCACCAGCC3019 CCTCGCAGGA TGGGGGTTTC AATGTGAAAC ATCTGCCAGT TTTGTTTTGT TTTTTTAATG3079 CTGCTTTGTC CAGGTGTCCA AACATCCATC ATTTGGGGTG GTCTGTTTTA CAGAGTAAAG3139 GAGGCGGTGA AGGGACGTCA GCTAGTGTGT AGAGCCAAGG GGAGACAGCT AGGATTCTCG3199 CCTAGCTGAA CCAAGGTGTA AAATAGAAGA CACGCTCC 3237 853 amino acidsamino acid linear protein unknown 6 Met Ala Val Thr Ser His His Met IlePro Val Met Val Val Leu Met 1 5 10 15 Ser Ala Cys Leu Ala Thr Ala GlyPro Glu Pro Ser Thr Arg Cys Glu 20 25 30 Leu Ser Pro Ile Asn Ala Ser HisPro Val Gln Ala Leu Met Glu Ser 35 40 45 Phe Thr Val Leu Ser Gly Cys AlaSer Arg Gly Thr Thr Gly Leu Pro 50 55 60 Arg Glu Val His Val Leu Asn LeuArg Ser Thr Asp Gln Gly Pro Gly 65 70 75 80 Gln Arg Gln Arg Glu Val ThrLeu His Leu Asn Pro Ile Ala Ser Val 85 90 95 His Thr His His Lys Pro IleVal Phe Leu Leu Asn Ser Pro Gln Pro 100 105 110 Leu Val Trp His Leu LysThr Glu Arg Leu Ala Ala Gly Val Pro Arg 115 120 125 Leu Phe Leu Val SerGlu Gly Ser Val Val Gln Phe Pro Ser Gly Asn 130 135 140 Phe Ser Leu ThrAla Glu Thr Glu Glu Arg Asn Phe Pro Gln Glu Asn 145 150 155 160 Glu HisLeu Val Arg Trp Ala Gln Lys Glu Tyr Gly Ala Val Thr Ser 165 170 175 PheThr Glu Leu Lys Ile Ala Arg Asn Ile Tyr Ile Lys Val Gly Glu 180 185 190Asp Gln Val Phe Pro Pro Thr Cys Asn Ile Gly Lys Asn Phe Leu Ser 195 200205 Leu Asn Tyr Leu Ala Glu Tyr Leu Gln Pro Lys Ala Ala Glu Gly Cys 210215 220 Val Leu Pro Ser Gln Pro His Glu Lys Glu Val His Ile Ile Glu Leu225 230 235 240 Ile Thr Pro Ser Ser Asn Pro Tyr Ser Ala Phe Gln Val AspIle Ile 245 250 255 Val Asp Ile Arg Pro Ala Gln Glu Asp Pro Glu Val ValLys Asn Leu 260 265 270 Val Leu Ile Leu Lys Cys Lys Lys Ser Val Asn TrpVal Ile Lys Ser 275 280 285 Phe Asp Val Lys Gly Asn Leu Lys Val Ile AlaPro Asn Ser Ile Gly 290 295 300 Phe Gly Lys Glu Ser Glu Arg Ser Met ThrMet Thr Lys Leu Val Arg 305 310 315 320 Asp Asp Ile Pro Ser Thr Gln GluAsn Leu Met Lys Trp Ala Leu Asp 325 330 335 Asn Gly Tyr Arg Pro Val ThrSer Tyr Thr Met Ala Pro Val Ala Asn 340 345 350 Arg Phe His Leu Arg LeuGlu Asn Asn Glu Glu Met Arg Asp Glu Glu 355 360 365 Val His Thr Ile ProPro Glu Leu Arg Ile Leu Leu Asp Pro Asp His 370 375 380 Pro Pro Ala LeuAsp Asn Pro Leu Phe Pro Gly Glu Gly Ser Pro Asn 385 390 395 400 Gly GlyLeu Pro Phe Pro Phe Pro Asp Ile Pro Arg Arg Gly Trp Lys 405 410 415 GluGly Glu Asp Arg Ile Pro Arg Pro Lys Gln Pro Ile Val Pro Ser 420 425 430Val Gln Leu Leu Pro Asp His Arg Glu Pro Glu Glu Val Gln Gly Gly 435 440445 Val Asp Ile Ala Leu Ser Val Lys Cys Asp His Glu Lys Met Val Val 450455 460 Ala Val Asp Lys Asp Ser Phe Gln Thr Asn Gly Tyr Ser Gly Met Glu465 470 475 480 Leu Thr Leu Leu Asp Pro Ser Cys Lys Ala Lys Met Asn GlyThr His 485 490 495 Phe Val Leu Glu Ser Pro Leu Asn Gly Cys Gly Thr ArgHis Arg Arg 500 505 510 Ser Thr Pro Asp Gly Val Val Tyr Tyr Asn Ser IleVal Val Gln Ala 515 520 525 Pro Ser Pro Gly Asp Ser Ser Gly Trp Pro AspGly Tyr Glu Asp Leu 530 535 540 Glu Ser Gly Asp Asn Gly Phe Pro Gly AspGly Asp Glu Gly Glu Thr 545 550 555 560 Ala Pro Leu Ser Arg Ala Gly ValVal Val Phe Asn Cys Ser Leu Arg 565 570 575 Gln Leu Arg Asn Pro Ser GlyPhe Gln Gly Gln Leu Asp Gly Asn Ala 580 585 590 Thr Phe Asn Met Glu LeuTyr Asn Thr Asp Leu Phe Leu Val Pro Ser 595 600 605 Pro Gly Val Phe SerVal Ala Glu Asn Glu His Val Tyr Val Glu Val 610 615 620 Ser Val Thr LysAla Asp Gln Asp Leu Gly Phe Ala Ile Gln Thr Cys 625 630 635 640 Phe LeuSer Pro Tyr Ser Asn Pro Asp Arg Met Ser Asp Tyr Thr Ile 645 650 655 IleGlu Asn Ile Cys Pro Lys Asp Asp Ser Val Lys Phe Tyr Ser Ser 660 665 670Lys Arg Val His Phe Pro Ile Pro His Ala Glu Val Asp Lys Lys Arg 675 680685 Phe Ser Phe Leu Phe Lys Ser Val Phe Asn Thr Ser Leu Leu Phe Leu 690695 700 His Cys Glu Leu Thr Leu Cys Ser Arg Lys Lys Gly Ser Leu Lys Leu705 710 715 720 Pro Arg Cys Val Thr Pro Asp Asp Ala Cys Thr Ser Leu AspAla Thr 725 730 735 Met Ile Trp Thr Met Met Gln Asn Lys Lys Thr Phe ThrLys Pro Leu 740 745 750 Ala Val Val Leu Gln Val Asp Tyr Lys Glu Asn ValPro Ser Thr Lys 755 760 765 Asp Ser Ser Pro Ile Pro Pro Pro Pro Pro GlnIle Phe His Gly Leu 770 775 780 Asp Thr Leu Thr Val Met Gly Ile Ala PheAla Ala Phe Val Ile Gly 785 790 795 800 Ala Leu Leu Thr Gly Ala Leu TrpTyr Ile Tyr Ser His Thr Gly Glu 805 810 815 Thr Ala Arg Arg Gln Gln ValPro Thr Ser Pro Pro Ala Ser Glu Asn 820 825 830 Ser Ser Ala Ala His SerIle Gly Ser Thr Gln Ser Thr Pro Cys Ser 835 840 845 Ser Ser Ser Thr Ala850 2090 base pairs nucleic acid double linear DNA (genomic) unknown CDS336..2038 7 GTTGGCGAGG AGTTTCCTGT TTCCCCCGCA GCGCTGAGTT GAAGTTGAGTGAGTCACTCG 60 CGCGCACGGA GCGACGACAC CCCCGCGCGT GCACCCGCTC GGGACAGGAGCCGGACTCCT 120 GTGCAGCTTC CCTCGGCCGC CGGGGGCCTC CCCGCGCCTC GCCGGCCTCCAGGCCCCTCC 180 TGGCTGGCGA GCGGGCGCCA CATCTGGCCC GCACATCTGC GCTGCCGGCCCGGCGCGGGG 240 TCCGGAGAGG GCGCGGCGCG GAGCGCAGCC AGGGGTCCGG GAAGGCGCCGTCCGTGCGCT 300 GGGGGCTCGG TCTATGACGA GCAGCGGGGT CTGCC ATG GGT CGG GGGCTG CTC 353 Met Gly Arg Gly Leu Leu 855 AGG GGC CTG TGG CCG CTG CAC ATCGTC CTG TGG ACG CGT ATC GCC AGC 401 Arg Gly Leu Trp Pro Leu His Ile ValLeu Trp Thr Arg Ile Ala Ser 860 865 870 875 ACG ATC CCA CCG CAC GTT CAGAAG TCG GTT AAT AAC GAC ATG ATA GTC 449 Thr Ile Pro Pro His Val Gln LysSer Val Asn Asn Asp Met Ile Val 880 885 890 ACT GAC AAC AAC GGT GCA GTCAAG TTT CCA CAA CTG TGT AAA TTT TGT 497 Thr Asp Asn Asn Gly Ala Val LysPhe Pro Gln Leu Cys Lys Phe Cys 895 900 905 GAT GTG AGA TTT TCC ACC TGTGAC AAC CAG AAA TCC TGC ATG AGC AAC 545 Asp Val Arg Phe Ser Thr Cys AspAsn Gln Lys Ser Cys Met Ser Asn 910 915 920 TGC AGC ATC ACC TCC ATC TGTGAG AAG CCA CAG GAA GTC TGT GTG GCT 593 Cys Ser Ile Thr Ser Ile Cys GluLys Pro Gln Glu Val Cys Val Ala 925 930 935 GTA TGG AGA AAG AAT GAC GAGAAC ATA ACA CTA GAG ACA GTT TGC CAT 641 Val Trp Arg Lys Asn Asp Glu AsnIle Thr Leu Glu Thr Val Cys His 940 945 950 955 GAC CCC AAG CTC CCC TACCAT GAC TTT ATT CTG GAA GAT GCT GCT TCT 689 Asp Pro Lys Leu Pro Tyr HisAsp Phe Ile Leu Glu Asp Ala Ala Ser 960 965 970 CCA AAG TGC ATT ATG AAGGAA AAA AAA AAG CCT GGT GAG ACT TTC TTC 737 Pro Lys Cys Ile Met Lys GluLys Lys Lys Pro Gly Glu Thr Phe Phe 975 980 985 ATG TGT TCC TGT AGC TCTGAT GAG TGC AAT GAC AAC ATC ATC TTC TCA 785 Met Cys Ser Cys Ser Ser AspGlu Cys Asn Asp Asn Ile Ile Phe Ser 990 995 1000 GAA GAA TAT AAC ACC AGCAAT CCT GAC TTG TTG CTA GTC ATA TTT CAA 833 Glu Glu Tyr Asn Thr Ser AsnPro Asp Leu Leu Leu Val Ile Phe Gln 1005 1010 1015 GTG ACA GGC ATC AGCCTC CTG CCA CCA CTG GGA GTT GCC ATA TCT GTC 881 Val Thr Gly Ile Ser LeuLeu Pro Pro Leu Gly Val Ala Ile Ser Val 1020 1025 1030 1035 ATC ATC ATCTTC TAC TGC TAC CGC GTT AAC CGG CAG CAG AAG CTG AGT 929 Ile Ile Ile PheTyr Cys Tyr Arg Val Asn Arg Gln Gln Lys Leu Ser 1040 1045 1050 TCA ACCTGG GAA ACC GGC AAG ACG CGG AAG CTC ATG GAG TTC AGC GAG 977 Ser Thr TrpGlu Thr Gly Lys Thr Arg Lys Leu Met Glu Phe Ser Glu 1055 1060 1065 CACTGT GCC ATC ATC CTG GAA GAT GAC CGC TCT GAC ATC AGC TCC ACG 1025 His CysAla Ile Ile Leu Glu Asp Asp Arg Ser Asp Ile Ser Ser Thr 1070 1075 1080TGT GCC AAC AAC ATC AAC CAC AAC ACA GAG CTG CTG CCC ATT GAG CTG 1073 CysAla Asn Asn Ile Asn His Asn Thr Glu Leu Leu Pro Ile Glu Leu 1085 10901095 GAC ACC CTG GTG GGG AAA GGT CGC TTT GCT GAG GTC TAT AAG GCC AAG1121 Asp Thr Leu Val Gly Lys Gly Arg Phe Ala Glu Val Tyr Lys Ala Lys1100 1105 1110 1115 CTG AAG CAG AAC ACT TCA GAG CAG TTT GAG ACA GTG GCAGTC AAG ATC 1169 Leu Lys Gln Asn Thr Ser Glu Gln Phe Glu Thr Val Ala ValLys Ile 1120 1125 1130 TTT CCC TAT GAG GAG TAT GCC TCT TGG AAG ACA GAGAAG GAC ATC TTC 1217 Phe Pro Tyr Glu Glu Tyr Ala Ser Trp Lys Thr Glu LysAsp Ile Phe 1135 1140 1145 TCA GAC ATC AAT CTG AAG CAT GAG AAC ATA CTCCAG TTC CTG ACG GCT 1265 Ser Asp Ile Asn Leu Lys His Glu Asn Ile Leu GlnPhe Leu Thr Ala 1150 1155 1160 GAG GAG CGG AAG ACG GAG TTG GGG AAA CAATAC TGG CTG ATC ACC GCC 1313 Glu Glu Arg Lys Thr Glu Leu Gly Lys Gln TyrTrp Leu Ile Thr Ala 1165 1170 1175 TTC CAC GCC AAG GGC AAC CTA CAG GAGTAC CTG ACG CGG CAT GTC ATC 1361 Phe His Ala Lys Gly Asn Leu Gln Glu TyrLeu Thr Arg His Val Ile 1180 1185 1190 1195 AGC TGG GAG GAC CTG CGC AAGCTG GGC AGC TCC CTC GCC CGG GGG ATT 1409 Ser Trp Glu Asp Leu Arg Lys LeuGly Ser Ser Leu Ala Arg Gly Ile 1200 1205 1210 GCT CAC CTC CAC AGT GATCAC ACT CCA TGT GGG AGG CCC AAG ATG CCC 1457 Ala His Leu His Ser Asp HisThr Pro Cys Gly Arg Pro Lys Met Pro 1215 1220 1225 ATC GTG CAC AGG GACCTC AAG AGC TCC AAT ATC CTC GTG AAG AAC GAC 1505 Ile Val His Arg Asp LeuLys Ser Ser Asn Ile Leu Val Lys Asn Asp 1230 1235 1240 CTA ACC TGC TGCCTG TGT GAC TTT GGG CTT TCC CTG CGT CTG GAC CCT 1553 Leu Thr Cys Cys LeuCys Asp Phe Gly Leu Ser Leu Arg Leu Asp Pro 1245 1250 1255 ACT CTG TCTGTG GAT GAC CTG GCT AAC AGT GGG CAG GTG GGA ACT GCA 1601 Thr Leu Ser ValAsp Asp Leu Ala Asn Ser Gly Gln Val Gly Thr Ala 1260 1265 1270 1275 AGATAC ATG GCT CCA GAA GTC CTA GAA TCC AGG ATG AAT TTG GAG AAT 1649 Arg TyrMet Ala Pro Glu Val Leu Glu Ser Arg Met Asn Leu Glu Asn 1280 1285 1290GCT GAG TCC TTC AAG CAG ACC GAT GTC TAC TCC ATG GCT CTG GTG CTC 1697 AlaGlu Ser Phe Lys Gln Thr Asp Val Tyr Ser Met Ala Leu Val Leu 1295 13001305 TGG GAA ATG ACA TCT CGC TGT AAT GCA GTG GGA GAA GTA AAA GAT TAT1745 Trp Glu Met Thr Ser Arg Cys Asn Ala Val Gly Glu Val Lys Asp Tyr1310 1315 1320 GAG CCT CCA TTT GGT TCC AAG GTG CGG GAG CAC CCC TGT GTCGAA AGC 1793 Glu Pro Pro Phe Gly Ser Lys Val Arg Glu His Pro Cys Val GluSer 1325 1330 1335 ATG AAG GAC AAC GTG TTG AGA GAT CGA GGG CGA CCA GAAATT CCC AGC 1841 Met Lys Asp Asn Val Leu Arg Asp Arg Gly Arg Pro Glu IlePro Ser 1340 1345 1350 1355 TTC TGG CTC AAC CAC CAG GGC ATC CAG ATG GTGTGT GAG ACG TTG ACT 1889 Phe Trp Leu Asn His Gln Gly Ile Gln Met Val CysGlu Thr Leu Thr 1360 1365 1370 GAG TGC TGG GAC CAC GAC CCA GAG GCC CGTCTC ACA GCC CAG TGT GTG 1937 Glu Cys Trp Asp His Asp Pro Glu Ala Arg LeuThr Ala Gln Cys Val 1375 1380 1385 GCA GAA CGC TTC AGT GAG CTG GAG CATCTG GAC AGG CTC TCG GGG AGG 1985 Ala Glu Arg Phe Ser Glu Leu Glu His LeuAsp Arg Leu Ser Gly Arg 1390 1395 1400 AGC TGC TCG GAG GAG AAG ATT CCTGAA GAC GGC TCC CTA AAC ACT ACC 2033 Ser Cys Ser Glu Glu Lys Ile Pro GluAsp Gly Ser Leu Asn Thr Thr 1405 1410 1415 AAA TA GCTCTTATGG GGCAGGCTGGGCATGTCCAA AGAGGCTGCC CCTCTCACCA 2088 Lys 1420 AA 2090 567 amino acidsamino acid linear protein unknown 8 Met Gly Arg Gly Leu Leu Arg Gly LeuTrp Pro Leu His Ile Val Leu 1 5 10 15 Trp Thr Arg Ile Ala Ser Thr IlePro Pro His Val Gln Lys Ser Val 20 25 30 Asn Asn Asp Met Ile Val Thr AspAsn Asn Gly Ala Val Lys Phe Pro 35 40 45 Gln Leu Cys Lys Phe Cys Asp ValArg Phe Ser Thr Cys Asp Asn Gln 50 55 60 Lys Ser Cys Met Ser Asn Cys SerIle Thr Ser Ile Cys Glu Lys Pro 65 70 75 80 Gln Glu Val Cys Val Ala ValTrp Arg Lys Asn Asp Glu Asn Ile Thr 85 90 95 Leu Glu Thr Val Cys His AspPro Lys Leu Pro Tyr His Asp Phe Ile 100 105 110 Leu Glu Asp Ala Ala SerPro Lys Cys Ile Met Lys Glu Lys Lys Lys 115 120 125 Pro Gly Glu Thr PhePhe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn 130 135 140 Asp Asn Ile IlePhe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp Leu 145 150 155 160 Leu LeuVal Ile Phe Gln Val Thr Gly Ile Ser Leu Leu Pro Pro Leu 165 170 175 GlyVal Ala Ile Ser Val Ile Ile Ile Phe Tyr Cys Tyr Arg Val Asn 180 185 190Arg Gln Gln Lys Leu Ser Ser Thr Trp Glu Thr Gly Lys Thr Arg Lys 195 200205 Leu Met Glu Phe Ser Glu His Cys Ala Ile Ile Leu Glu Asp Asp Arg 210215 220 Ser Asp Ile Ser Ser Thr Cys Ala Asn Asn Ile Asn His Asn Thr Glu225 230 235 240 Leu Leu Pro Ile Glu Leu Asp Thr Leu Val Gly Lys Gly ArgPhe Ala 245 250 255 Glu Val Tyr Lys Ala Lys Leu Lys Gln Asn Thr Ser GluGln Phe Glu 260 265 270 Thr Val Ala Val Lys Ile Phe Pro Tyr Glu Glu TyrAla Ser Trp Lys 275 280 285 Thr Glu Lys Asp Ile Phe Ser Asp Ile Asn LeuLys His Glu Asn Ile 290 295 300 Leu Gln Phe Leu Thr Ala Glu Glu Arg LysThr Glu Leu Gly Lys Gln 305 310 315 320 Tyr Trp Leu Ile Thr Ala Phe HisAla Lys Gly Asn Leu Gln Glu Tyr 325 330 335 Leu Thr Arg His Val Ile SerTrp Glu Asp Leu Arg Lys Leu Gly Ser 340 345 350 Ser Leu Ala Arg Gly IleAla His Leu His Ser Asp His Thr Pro Cys 355 360 365 Gly Arg Pro Lys MetPro Ile Val His Arg Asp Leu Lys Ser Ser Asn 370 375 380 Ile Leu Val LysAsn Asp Leu Thr Cys Cys Leu Cys Asp Phe Gly Leu 385 390 395 400 Ser LeuArg Leu Asp Pro Thr Leu Ser Val Asp Asp Leu Ala Asn Ser 405 410 415 GlyGln Val Gly Thr Ala Arg Tyr Met Ala Pro Glu Val Leu Glu Ser 420 425 430Arg Met Asn Leu Glu Asn Ala Glu Ser Phe Lys Gln Thr Asp Val Tyr 435 440445 Ser Met Ala Leu Val Leu Trp Glu Met Thr Ser Arg Cys Asn Ala Val 450455 460 Gly Glu Val Lys Asp Tyr Glu Pro Pro Phe Gly Ser Lys Val Arg Glu465 470 475 480 His Pro Cys Val Glu Ser Met Lys Asp Asn Val Leu Arg AspArg Gly 485 490 495 Arg Pro Glu Ile Pro Ser Phe Trp Leu Asn His Gln GlyIle Gln Met 500 505 510 Val Cys Glu Thr Leu Thr Glu Cys Trp Asp His AspPro Glu Ala Arg 515 520 525 Leu Thr Ala Gln Cys Val Ala Glu Arg Phe SerGlu Leu Glu His Leu 530 535 540 Asp Arg Leu Ser Gly Arg Ser Cys Ser GluGlu Lys Ile Pro Glu Asp 545 550 555 560 Gly Ser Leu Asn Thr Thr Lys 565

What is claimed is:
 1. An antibody preparation consisting of antibodieswhich specifically recognize mammalian TGF-β type II receptor.
 2. Theantibody preparation according to claim 1, wherein the TGF-β type IIreceptor is the human TGF-β type II receptor.
 3. The antibodypreparation according to claim 1 or 2, wherein the antibodies aremonoclonal antibodies.
 4. An antibody preparation consisting ofantibodies which specifically recognize a polypeptide comprising anamino acid sequence of a mammalian TGF-β type III receptor proteinselected from the group consisting of: a) the amino acid sequenceencoded by a cDNA insert contained in the plasmid deposited under ATCCaccession number 75127, and b) the amino acid sequence set forth in SEQID NO:6.
 5. The antibody preparation according to claim 4, wherein thepolypeptide comprises the amino acid sequence encoded by a cDNA insertcontained in the plasmid deposited under ATCC accession number
 75127. 6.The antibody preparation according to claim 4 or 5, wherein theantibodies are monoclonal antibodies.